Comparison of the soluble and membrane-bound forms of the puromycin-sensitive enkephalin-degrading aminopeptidases from rat.
Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.
Dyer
S H
SH
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
Slaughter
C A
CA
Orth
K
K
Moomaw
C R
CR
Hersh
L B
LB
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000502:physiology; D001921:Brain / Q000201:enzymology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D004745:Enkephalins / Q000378:metabolism*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010449:Peptide Mapping; D011691:Puromycin / Q000494:pharmacology*; D051381:Rats; D012995:Solubility
D000906:Antibodies; D004745:Enkephalins; D011691:Puromycin; D000626:Aminopeptidases